The present invention relates to methods for sterilizing preparations of digestive enzymes to reduce the level of one or more active biological contaminants or pathogens therein, such as viruses, bacteria (including inter- and intracellular bacteria, such as mycoplasmas, ureaplasmas, nanobacteria, chlamydia, rickettsias), yeasts, molds, fungi, prions or similar agents responsible, alone or in combination, for TSEs and/or single or multicellular parasites. The present invention particularly relates to methods of sterilizing preparations of digestive enzymes, such as trypsin, xcex1-galactosidase and iduronate 2-sulfatase, with irradiation.
The principal foods upon which an organism, such as a human, survives can be broadly categorized as carbohydrates, fats and proteins. These substances, however, are useless as nutrients without the process of digestion to break down foods.
Digestion of carbohydrates begins in the mouth and stomach. Saliva contains the enzyme ptyalin (an alpha-amylase), which hydrolyses starch into maltose and other small polymers of glucose. The pancreatic alpha-amylase is similar to the salivary ptyalin, but several times as powerful. Therefore, soon after chyme empties into the duodenum and mixes with pancreatic juice, virtually all of the starches are converted into disaccharides and small glucose polymers. These disaccharides and small glucose polymers are hydrolysed into monosaccharides by intestinal epithelial enzymes.
Digestion of proteins begins in the stomach. The enzyme pepsin, which is produced in the stomach, digests collagen, a major constituent of the intercellular connective tissue of meats. This enzymatic reaction is essential so that other digestive enzymes can penetrate meats and digest the cellular proteins. Consequently, in people who lack peptic activity in the stomach, the ingested meats are not well penetrated by these other digestive enzymes and so are poorly absorbed.
Most protein digestion results from the actions of the pancreatic proteolytic enzymes. Proteins leaving the stomach in the form of proteoses, peptones and large polypeptides are digested into dipeptides, tripeptides and the like by pancreatic proteolytic enzymes or polypeptidases. Trypsin and chymotrypsin split protein molecules into smaller polypeptides at specific peptide linkages, while carboxypolypeptidase cleaves amino acids from the carboxyl ends of polypeptides. Proelastase gives rise to elastase, which in turn digests the elastin fibers that hold together most meat.
Further digestion of polypeptides takes place in the intestinal lumen. Aminopolypeptidase and several polypeptidases split large polypeptides into dipeptides, tripeptides and amino acids, which are transported into the enterocytes that line the intestinal villi. Inside the enterocytes, other polypeptidases split the remaining peptides into their constituent amino acids, which then enter the blood.
Digestion of fats first requires emulsification by bile acids and lecithin, which increase the surface area of the fats up to 1000-fold. Because lipases are water-soluble digestive enzymes that can bind only on the surface of a fat globule, this emulsification process is important for the complete digestion of fat. The most important digestive enzyme in the digestion of triglycerides is pancreatic lipase, which breaks these down into free fatty acids and 2-monoglycerides. After these free fatty acids and monoglycerides enter the enterocytes, they are generally recombined into new triglyerides. A few monoglycerides, however, are further digested by intracellular lipases into free fatty acids.
Digestion therefore continues after the breakdown and uptake of nutrients into the various cells of the body. Intracellular enzymes, such as intracellular lipases, are involved in the uptake, breakdown, transport, storage, release, metabolism and catabolism of nutrients into forms required and useable by the cell(s) of an organism at various places and times. This includes storage of lipids and their metabolism into energy sources as well as their catabolism and synthesis into other useful compounds. Digestion may also occur as a part of an organism""s normal process(es) of tissue generation and regeneration or repair of degraded, damaged or abnormal tissue(s) or molecules. It may also be a feature of or result from apoptosis, immune reactions, infections, neoplasms and other abnormal or disease states of an organism.
Preparations of digestive enzymes are therefore often provided therapeutically to humans and animals.
For example, in cases of pancreatitis and lack of pancreatic secretion, preparations of certain pancreatic enzymes, including combinations of lipase, protease and amylase (such as Creon(trademark), Cotazym(trademark), Donnazyme(trademark), Ku-Zyme(trademark) HP, Pancrease(trademark) and Pancrease(trademark) MT, Ultrase(trademark) and Ultrase(trademark) MT, Viokase(trademark), and Zymase(trademark)) and combinations of lipase, protease, amylase and cellulase (such as Ku-Zyme(trademark) and Kutrase(trademark)), are administered to ensure proper patient nutrition. The digestive enzymes of particular interest, for example in replacement therapy in humans and animals, therefore include pancreatic digestive enzymes, such as trypsin and chymotrypsin, and functional mutants, variants and derivatives thereof.
Trypsin is an enzyme that acts to degrade protein; it is often referred to as a digestive enzyme, or proteinase. In the digestive process, trypsin acts with the other proteinases to break down dietary protein molecules to their component peptides and amino acids. Trypsin continues the process of digestion (begun in the stomach) in the small intestine where a slightly alkaline environment (about pH 8) promotes its maximal enzymatic activity. Trypsin, produced in an inactive form by the pancreas, is remarkably similar in chemical composition and in structure to the other chief pancreatic proteinase, chymotrypsin. Both enzymes also appear to have similar mechanisms of action; residues of histidine and serine are found in the active sites of both. The chief difference between the two molecules seems to be in their specificity, that is, each is active only against the peptide bonds in protein molecules that have carboxyl groups donated by certain amino acids. For trypsin these amino acids are arginine and lysine, for chymotrypsin they are tyrosine, phenylalanine, tryptophan, methionine, and leucine. Trypsin is the most discriminating of all the digestive enzymes in terms of the restricted number of chemical bonds that it will attack.
Preparations of other digestive enzymes, such as glycosidases, are likewise administered therapeutically to human patients. For example, Fabry disease is an X-linked recessive glycolipid storage disorder caused by a deficiency of the lysosomal enzyme xcex1-galactosidase A. Clinical manifestations of Fabry disease included recurrent episodes severe pain and progressive renal, cardiac and cerebrovascular deterioration with death usually occurring in the fourth to sixth decade of life. Enzyme replacement therapy by infusion of a preparation of xcex1-galactosidase A has been tested and found to be a promising potential therapy for this condition (Schiffmann, et al, xe2x80x9cEnzyme Replacement Therapy in Fabry Disease: A Randomized Controlled Trial.xe2x80x9d JAMA, Jun. 6, 2001, Vol. 285, No. 21, pp. 2743-2749.).
Glycogen Storage Disease Type II (also known as Acid Maltase Deficiency or Pompe Disease) is another genetically transmitted storage disorder. In GSD-II, the patient suffers from a deficiency of acid maltase enzyme, which breaks down glycogen in muscle cells. Clinical manifestations of GSD-II include progressive muscle weakness due to a build up of glycogen in muscle tissues, eventually resulting in respiratory and/or cardiac failure. Preparations of glycosidases, or functional mutants or variants or derivatives thereof, are therefore also of particular interest for therapeutic use.
Niemann-Pick Disease is also a genetically transmitted metabolic disorder in which harmful quantities of a fatty substance, sphingomyelin, accumulate in the spleen, liver, lungs, bone marrow and brain. Patients suffer from a deficiency of sphingomyelinases, which initiates the biodegradation of sphinogmyelin. Clinical manifestations include enlargement of the spleen and liver, and frequently results in death, particularly for pediatric patients.
Gaucher""s Disease is a somewhat-similar genetically transmitted disorder, in which harmful quantities of another fatty substance, glucocerebroside, accumulate in the spleen, liver, lungs, bone marrow and brain. Patients suffer from a deficiency in xcex2-glucocerebrosidase, which catalyzes the first step in the biodegradation of glucocerebroside, which arises from the biodegradation of old red and white blood cells. Clinical manifestations include enlargement of the spleen and liver, low blood platelets, fatigue and, in certain forms, progressive brain damage. Enzyme replacement therapy by infusion of a preparation of a modified form of glucocerebrosidase, known as algucerase (Ceredase(trademark)) has been tested and found to be a promising potential therapy for this condition (Barton, et al., xe2x80x9cReplacement Therapy for Enzyme Deficiency: Macrophage-targeted Glucocerebrosidase for Gaucher""s Disease.xe2x80x9d New Engl. J. Med., May 23, 1991.).
Mucopolysaccharidoses are a group of inherited metabolic disorders caused by a deficiency in the lysosomal enzymes needed to break down mucopolysaccharides, long chains of sugar molecules used to build connective tissue and organs in the body. A deficiency in one or more of these enzymes cases a build up of excess amount in the body, causing progressive damage and eventual death. Among these disorders are Hurler, Scheie and Hurler/Scheie syndromes (the most severe form, occurs in infancy with death resulting before age 10 years, symptoms include clouding of the cornea and progressive physical and mental disability, caused by a deficiency in xcex1-L-iduronidase), Hunter syndrome (affects juveniles with death usually resulting by age 15 years, symptoms include joint stiffness, mental deterioration, dwarfing and progressive deafness, caused by a deficiency in iduronate-2-sulfatase), Sanfillipo syndrome (death usually occurs by late teens, symptoms include progressive dementia and mental deterioration in childhood, caused by a deficiency in heparan N-sulfatase, xcex1-N-acetylglucosaminadase, acetyl-CoA-glucosaminide acetyltransferase and/or N-acetylglucosamine-6-sulfatase), Morquio syndrome (appears in infancy, symptoms include severe dwarfing and corneal clouding, cardiac or respiratory disease may cause death in third or fourth decade of like, caused by a deficiency in galactosamine-6-sulfatase and/or xcex2-galactosidase), Maroteauz-Lamy syndrome (resembles Hurler syndrome, onset in infancy, but no mental disability, death usually occurs in second or third decade of life, caused by a deficiency in arylsulfatase B), and Sly disease (symptoms include corneal clouding, skeletal irregularities, and enlargement of the liver and spleen, caused by a deficiency in xcex2-glucuronidase). Hunter syndrome is particularly linked to a deficiency in iduronate-2-sulfatase, which catalyzes the breakdown of heparan sulfate and dermatan sulfate, and it has been suggested that this condition can be treated by administration of variant forms of the enzyme (U.S. Pat. No. 6,153,188). The digestive of particular interest, for example in therapy in humans and animals, therefore also include iduronate-2-sulfatase and functional mutants, variants and derivatives thereof.
Multiple Sulfatase Deficiency (also known as Disorder of Confication 13 or Mucosulfatidosis) is another hereditary metabolic disorder characterized by impairment of all known sulfatase enzymes (including arylsulfatases A, B and C, two steroid sulfatases and four other sulfatases). Clinical manifestations include coarse facial features, deafness, an enlarged liver and spleen, abnormalities of the skeleton (including lumbar kyphosis) and dry, scaly skin (ichthyosis).
Similarly, preparations of digestive enzymes are administered to humans and animals to improve nutrition.
For example, in cases of lactose intolerance, preparations of lactase (such as Lactaid(trademark)) are administered to humans in need thereof. Lactose intolerance is characterized by gastrointestinal discomfort, including gas, bloating, crampls and diarrhea, after the consumption of milk or milk-containing products. The digestive enzymes of particular interest, for example in therapy in humans and animals, therefore also include lactase and functional mutants, variants and derivatives thereof.
Likewise, preparations of galactosidases (such as Beano(trademark) or Nutritek(trademark) Alpha Galactosidase) are administered to humans in need thereof. Such products improve digestion of sugars found in foods including legumes and cruciferous vegetables and reduce effects generally associated with the foods, such as gas and bloating.
Preparations of digestive enzymes that are prepared for human, veterinary, diagnostic and/or experimental use may contain unwanted and potentially dangerous biological contaminants or pathogens, such as viruses, bacteria (including inter- and intracellular bacteria, such as mycoplasmas, ureaplasmas, nanobacteria, chlamydia, rickettsias), yeasts, molds, fungi, prions or similar agents responsible, alone or in combination, for TSEs and/or single or multicellular parasites. Consequently, it is of utmost importance that any biological contaminant in the preparation be inactivated before the product is used. This is especially critical when the preparation is to be administered directly to a patient, for example in human therapy corrected or treated by intravenous, intramuscular or other forms of injection. This is also critical for the various preparations that are prepared in media or via culture of cells or recombinant cells which contain various types of plasma and/or plasma derivatives or other biological materials or are used to prepare biological materials for human use and which may be subject to mycoplasma, prion, bacterial, viral and/or other biological contaminants or pathogens.
Most procedures for producing preparations of digestive enzymes have involved methods that screen or test the preparation for one or more particular biological contaminants or pathogens rather than removal or inactivation of the contaminant(s) and/or pathogen(s) from the preparation. Preparations that test positive for a biological contaminant or pathogen are merely not used. Examples of screening procedures include the testing for a particular virus in human blood from blood donors. Such procedures, however, are not always reliable and are not able to detect the presence of certain viruses, particularly in very low numbers, and in the case of as yet unknown viruses or other contaminants or pathogens that may be in blood. This reduces the value or certainty of the test in view of the consequences associated with a false negative result. False negative results can be life threatening in certain cases, for example in the case of Acquired Immune Deficiency Syndrome (AIDS). Furthermore, in some instances it can take weeks, if not months, to determine whether or not the preparation is contaminated. Therefore, it would be desirable to apply techniques that would kill or inactivate biological contaminants and pathogens during and/or after manufacturing the preparation of digestive enzymes.
In conducting experiments to determine the ability of technologies to inactivate viruses, the actual viruses of concern are seldom utilized. This is a result of safety concerns for the workers conducting the tests, and the difficulty and expense associated with the containment facilities and waste disposal. In their place, model viruses of the same family and class are used.
In general, it is acknowledged that the most difficult viruses to inactivate are those with an outer shell made up of proteins, and that among these, the most difficult to inactivate are those of the smallest size. This has been shown to be true for gamma irradiation and most other forms of radiation as these viruses"" diminutive size is associated with a small genome. The magnitude of direct effects of radiation upon a molecule are directly proportional to the size of the molecule, that is the larger the target molecule, the greater the effect. As a corollary, it has been shown for gamma-irradiation that the smaller the viral genome, the higher the radiation dose required to inactive it.
Among the viruses of concern for both human and animal-derived preparations, the smallest, and thus most difficult to inactivate, belong to the family of Parvoviruses and the slightly larger protein-coated Hepatitis virus. In humans, the Parvovirus B19, and Hepatitis A are the agents of concern. In porcine-derived materials, the smallest corresponding virus is Porcine Parvovirus. Since this virus is harmless to humans, it is frequently chosen as a model virus for the human B19 Parvovirus. The demonstration of inactivation of this model parvovirus is considered adequate proof that the method employed will kill human B19 virus and Hepatitis A, and by extension, that it will also kill the larger and less hardy viruses such as HIV, CMV, Hepatitis B and C and others.
More recent efforts have focussed on methods to remove or inactivate contaminants in the products. Such methods include heat treating, filtration and the addition of chemical inactivants or sensitizers to the product.
Heat treatment requires that the product be heated to approximately 60xc2x0 C. for about 70 hours which can be damaging to sensitive products. In some instances, heat inactivation can actually destroy 50% or more of the biological activity of the product.
Filtration involves filtering the product in order to physically remove contaminants. Unfortunately, this method may also remove products that have a high molecular weight. Further, in certain cases, small viruses and similarly sized contaminants and pathogens, such as prions, may not be removed by the filter.
The procedure of chemical sensitization involves the addition of noxious agents which bind to the DNA/RNA of the virus and which are activated either by UV or other radiation. This radiation produces reactive intermediates and/or free radicals which bind to the DNA/RNA of the virus, break the chemical bonds in the backbone of the DNA/RNA, and/or cross-link or complex it in such a way that the virus can no longer replicate. This procedure requires that unbound sensitizer is washed from products since the sensitizers are toxic, if not mutagenic or carcinogenic, and cannot be administered to a patient.
Irradiating a product with gamma radiation is another method of sterilizing a product. Gamma radiation is effective in destroying viruses and bacteria when given in high total doses (Keathly et al, xe2x80x9cIs There Life After Irradiation? Part 2,xe2x80x9d BioPharm July-August, 1993, and Leitman, USe of Blood Cell Irradiation in the Prevention of Post Transfusion Graft-vs-Host Disease,xe2x80x9d Transfusion Science 10:219-239 (1989)). The published literature in this area, however, teaches that gamma radiation can be damaging to radiation sensitive products, such as blood, blood products, enzymes, protein and protein-containing products. In particular, it has been shown that high radiation doses are injurious to red cells, platelets and granulocytes (Leitman). U.S. Pat. No. 4,620,908 discloses that protein products must be frozen prior to irradiation in order to maintain the viability of the protein product. This patent concludes that xe2x80x9c[i]f the gamma irradiation were applied while the protein material was at, for example, ambient temperature, the material would be also completely destroyed, that is the activity of the material would be rendered so low as to be virtually ineffectivexe2x80x9d. Unfortunately, many sensitive biological materials, such as monoclonal antibodies (Mab), may lose viability and activity if subjected to freezing for irradiation purposes and then thawing prior to administration to a patient.
In view of the difficulties discussed above, there remains a need for methods of sterilizing preparations of one or more digestive enzymes that are effective for reducing the level of active biological contaminants or pathogens without an adverse effect on the preparation.
Accordingly, it is an object of the present invention to provide methods of sterilizing preparations of digestive enzymes by reducing the level of active biological contaminants or pathogens without adversely effecting the preparation. Other objects, features and advantages of the present invention will be set forth in the detailed description of preferred embodiments that follows, and in part will be apparent from the description or may be learned by practice of the invention. These objects and advantages of the invention will be realized and attained by the compositions and methods particularly pointed out in the written description and claims hereof.
In accordance with these and other objects, a first embodiment of the present invention is directed to a method for sterilizing a preparation of one or more digestive enzymes that is sensitive to radiation comprising irradiating the preparation of one or more digestive enzymes with radiation for a time effective to sterilize the material at a rate effective to sterilize the material and to protect the material from radiation.
Another embodiment of the present invention is directed to a method for sterilizing a preparation of one or more digestive enzymes that is sensitive to radiation comprising: (i) adding to a preparation of one or more digestive enzymes at least one stabilizer in an amount effective to protect the preparation of one or more digestive enzymes from radiation; and (ii) irradiating the preparation of one or more digestive enzymes with radiation at an effective rate for a time effective to sterilize the material.
Another embodiment of the present invention is directed to a method for sterilizing a preparation of one or more digestive enzymes that is sensitive to radiation comprising: (i) reducing the residual solvent content of a preparation of one or more digestive enzymes to a level effective to protect the preparation of one or more digestive enzymes from radiation; and (ii) irradiating the preparation of one or more digestive enzymes with radiation at an effective rate for a time effective to sterilize the preparation of one or more digestive enzymes.
Another embodiment of the present invention is directed to a method for sterilizing a preparation of one or more digestive enzymes that is sensitive to radiation comprising: (i) reducing the temperature of a preparation of one or more digestive enzymes to a level effective to protect the preparation of one or more digestive enzymes from radiation; and (ii) irradiating the preparation of one or more digestive enzymes with radiation at an effective rate for a time effective to sterilize the preparation of one or more digestive enzymes.
Another embodiment of the present invention is directed to a method for sterilizing a preparation of one or more digestive enzymes that is sensitive to radiation comprising: (i) applying to the preparation of one or more digestive enzymes a stabilizing process selected from the group consisting of: (a) reducing the residual solvent content of a preparation of one or more digestive enzymes, (b) adding to the preparation of one or more digestive enzymes at least one stabilizer, and (c) reducing the temperature of the preparation of one or more digestive enzymes; and (ii) irradiating the preparation of one or more digestive enzymes with radiation at an effective rate for a time effective to sterilize the preparation of one or more digestive enzymes, wherein the stabilizing process and the rate of irradiation are together effective to protect the preparation of one or more digestive enzymes from radiation.
Another embodiment of the present invention is directed to a method for sterilizing a preparation of one or more digestive enzymes that is sensitive to radiation comprising: (i) applying to the preparation of one or more digestive enzymes at least two stabilizing processes selected from the group consisting of: (a) reducing the residual solvent content of a preparation of one or more digestive enzymes, (b) adding to the preparation of one or more digestive enzymes at least one stabilizer, and (c) reducing the temperature of the preparation of one or more digestive enzymes; and (ii) irradiating the preparation of one or more digestive enzymes with radiation at an effective rate for a time effective to sterilize the preparation of one or more digestive enzymes, wherein the stabilizing processes may be performed in any order and are together effective to protect the preparation of one or more digestive enzymes from radiation.
The invention also provides a biological composition comprising at least one preparation of one or more digestive enzymes and a least one stabilizer in an amount effective to preserve the preparation of one or more digestive enzymes for its intended use following sterilization with radiation.
The invention also provides a biological composition comprising at least one preparation of one or more digestive enzymes in which the residual solvent content has been reduced to a level effective to preserve the preparation of one or more digestive enzymes for its intended use following sterilization with radiation.
The invention also provides a biological composition comprising at least one preparation of one or more digestive enzymes and at least one stabilizer in which the residual solvent content has been reduced and wherein the amount of stabilizer and level of residual solvent content are together effective to preserve the preparation of one or more digestive enzymes for its intended use following sterilization with radiation.
The invention also provides a biological composition comprising at least one preparation of one or more digestive enzymes wherein the total protein concentration of the preparation is effective to preserve the preparation of one or more digestive enzymes for its intended use following sterilization with radiation.